Achieving a deep understanding of RNA catalysis is important on several fronts, with relevance to early solution, the targeting and use of RNAs in potential therapeutic interventions, the function of modern day RNA enzymes and molecular machines, and the fundamental principles that underlie biological catalysis. More specifically, study of group I introns funded bythis grant have provided insights into possible mechanisms for the transition from an RNAto an RNPworld, into how RNAchaperones may solve conformational roadblocks to RNAfunction, into conformational transitions that may be utilized in RNA molecular machines, and into fundamental aspects of biological catalysis such as the use of binding energy to facilitate chemical transformations. Further, the ease of manipulation of RNA at the chemical and biophysical level, and perhaps its less compact and less interdigitated structure relative to protein enzymes, have allowed a dissection of active site interactions for the Tetrahymena ribozyme beyond that for any metallo-phosphotransferase. The proposed research constitutes a continuation of our goals of achieving an in-depth understanding of RNA catalysis with the above broad goals in mind. TheTetrahymena group I intron is used as the predominant system because the detailed information and understanding of the RNA in terms of structure and mechanism allow the questions to be asked at the deepest level. The fundamental understanding sought in this study is critical for a deep and thorough understanding of health related issues and problems, including optimizing the use of nucleic acids as therapeutics, understanding the behavior of RNA in cellular processes,which, in addition to providing important basic biological information, may help in manipulating RNAs as drug targets and developing therapies against RNA viruses such as HIV, and understanding disease states that originate from malfunctions at the RNA or RNA/protein level.